DSpace at VNU: The production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest

DSpace at VNU: The production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest tài...

Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 ORIGINAL ARTICLE Open Access The production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest Ziqing Gao1, Duong Van Hop2, Le Thi Hoang Yen2, Katsuhiko Ando3, Shuichi Hiyamuta4 and Ryuichiro Kondo1* Abstract Fusarium proliferatum NBRC109045 is a filamentous fungus isolated from Vietnamese forest due to high production of β-glucosidases Production of the enzyme was studied on varied carbon source based mediums The highest activity was obtained in medium containing 1% corn stover + 1% wheat bran (3.31 ± 0.14 U/ml) It is interesting to note that glucose (0.69 ± 0.02 U/ml) gave higher activity and just followed by cellobiose among the di- and mono-saccharides, which is generally regarded as a universal repressor of hydrolases We improved the zymogram method to prove that in response to various carbon sources, F proliferatum could express various β-glucosidases One of the β-glucosidases produced by F proliferatum growing in corn stover + wheat bran based medium was partially purified and proved to have high catalytic ability Keywords: Fusarium proliferatum, β-glucosidases, Differential expression, The translation elongation factor 1-α Introduction Biofuels derived from lignocellulosic biomass are emerging as promising alternatives to fossil fuels to meet the increasing global energy demands (Ragauskas et al 2006) One of the key steps in bioconversion process is the enzymatic hydrolysis of the cellulose polymers in the biomass to monomeric sugars that are subsequently fermented to ethanol (Percival et al 2006; Adsul et al 2007) The three main categories of players in cellulose hydrolysis are cellobiohydrolases (or exo-1, 4-β-glucanases) (EC 3.2.1.91), endo-1, 4-β-glucanases (EC 3.2.1.4), and β-glucosidases (EC 3.2.1.21) (Beguin and Aubert 1994) The endo-1, 4-β-glucanases randomly attack cellulose in amorphous zones and release oligomers The cellobiohydrolases liberate cellobiose from reducing and non-reducing ends And finally β-glucosidases hydrolyze the cellobiose and in some cases the cellooligosaccharides to glucose (Ryu 1980; Wood 1985) Cellulose polymers are degraded to glucose through sequential and cooperative actions of these enzymes Cellobiohydrolases * Correspondence: ryukondo@agr.kyushu-u.ac.jp Department of Agro-Environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan Full list of author information is available at the end of the article and endoglucanases are often inhibited by cellobiose, making β-glucosidases important in terms of avoiding decreased hydrolysis rates of cellulose over time due to cellobiose accumulation (Workman and Day 1982) Low efficiency and high costs associated with the enzymatic hydrolysis process present a major bottleneck in the production of ethanol from lignocellulosic feedstocks (Banerjee et al 2010) For the enzymatic conversion of biomass to fermentative sugar on a commercial scale, it is necessary to have all cellulolytic components at the optimal level Since β-glucosidases activity is low in many microbial preparations used usually for the saccharification process (Enari 1983) It is necessary to supply additional β-glucosidases to such reaction In order to optimize the use of different biomasses, it is important to identify new β-glucosidases with improved abilities on the specific biomasses as well as with improved abilities such as stability and high conversion rates β-Glucosidases have potential roles in various fields such as the food, pharmacology and cosmetic industries and also in the valorisation of some products, due to the properties of this enzyme to convert and to synthesize biomolecules of high added value (Esen 1993) There are hundreds of different β-glucosidic flavor precursors in plants, and their hydrolysis often enhances the quality of the beverages and foods © 2012 Gao et al.; licensee Springer This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 produced from them (Gϋnata 2003; Esen 2003) Aside from flavor enhancement, foods, feeds, and beverages may be improved nutritionally by release of vitamins, antioxidants, and other beneficial compounds from their glycosides (Opassiri et al 2004) Indeed, β-glucosidase can either degrade or synthesize small carbohydrate polymers, depending on particular experimental conditions (Crout and Vic 1998) The β-glucosidases can be arranged in three groups related to localization: intracellular, cell wall associated, and extracellular Primarily the extracellular β-glucosidases are of industrial interest (Soewnsen 2010) The number of fungal species on earth is estimated to 1.5 million of which as little as approximately 5% are known (Hawksworth 1991; 2001) So there is a statement that calls for all-out effort to unravel the potential of unknown species found in nature The identification and characterization of new fungal species are often encountered in literature Cuc Phuong Park and Ba Be Park is the old national one in Vietnam and boasts an engaging cultural and wildlife heritage and enchanting scenery Covered in a dense forest, these landscapes are rich and diverse tropical and subtropical species of microorganisms for wood and plant degradation In the present study, a potential β-glucosidases-producing fungus NBRC109045 was isolated from Ba Be national park and identified as Fusarium proliferatum Under optimized conditions, F proliferatum produces β-glucosidases with an activity of 3.3 U/ml based on pNPG as substrate and an activity of 426 U/ml based on cellobiose as substrate In this paper, we described ways that (a) isolating and screening microbes to produce considerable quantities of β-glucosidases; (b) modifying the method of zymogram to prove that different carbon sources direct varied β-glucosidases expression in F proliferatum; (c) assaying partial purification to prove high catalytic efficiency of β-glucosidase produced by F proliferatum growing in corn stover + wheat bran based medium Materials and methods Materials Unless specified otherwise, all chemicals were of analytical grade Solubilized crystalline cellulose was obtained from Kyokuto Seiyaku Co., Ltd, Japan Avicel [(R) RH101], 4-methylumbelliferyl-β-D-glucoside (MUG) and carboxymethyl cellulose (CMC) were products of Sigma Chemical Co., (St Louis, Mo, USA) Cellobiose, xylose, glucose, sucrose, galactose and maltose were purchased from Wako Pure Chemical Industries, Ltd, Japan 4-Nitrophenyl-β-D-glucopyranoside monohydrate (pNPG) was purchased from Tokyo Chemical Industry Co., Ltd, Japan Corn stover was collected from Yingkou city, Liaoning Province in China Wheat bran and bagasse were obtained from private companies Page of 13 Strains isolation Wood chip of Jatropha carcass, branch and leaves of J carcass, wood chip of Manihot esculenta, branch and leaves of M esculenta, coconut shell, sugarcane, and rice straw were used as lignocellulosic sources for degradation in Vietnamese National Park (Ba Be and Cuc Phuong) One month later, lignocellulosic sources were dug up All strains that would be screened were isolated from degraded biomass samples and washed soil collected Isolated strains were inoculated on solubilized crystalline cellulose (CC) plates and CMC plates to cultivate for two weeks (Deguchi et al 2007) The microbes that could grow on CC and CMC were picked up and inoculated onto malt extract agar (MEA) Screening of β-glucosidases-producing strains The first step of screening For primary screening, strains from MEA were plated on potato dextrose agar (PDA) medium in a 9-cm diameter Petri dish and incubated at 30°C for days Then the colonies were inoculated on β-glucosidases (EC 3.2.1.21) screening agar containing 1% of CMC, 0.5% of MUG, 1.5% of agar, and Mandels salts (Daenen et al 2008) The cultures were incubated at 30°C for days Then the plates were observed under UV light Colonies which showed fluorescence were sorted out It is because methylumbelliferyl (MU) which was released from MUG by β-glucosidases can emit fluorescence when induced by UV light The second step of screening For secondary screening, the mycelium of the βglucosidases-producing isolates obtained from the primary screening was transferred to a new PDA medium in a 9-cm diameter Petri dish and incubated at 30°C Once the fungus covered most of the PDA plate, agar plates with mycelium were transferred to a sterile blender containing 25 ml of sterile water and homogenized for 30 s Ten ml of the fungal homogenate was used to inoculate into β-glucosidases secondary screening medium containing 1% corn stover + 1% wheat bran in 100 ml, pH 5.0 Mandels salts medium with KH2PO4 g l-1, (NH4)2SO4 1.4 g l-1, urea 0.69 g l-1, CaCl2Á2H2O 0.3 g l-1, MgSO4Á7H2O 0.3 g l-1, and ml trace elements solution composing of MnSO4 1.6 g l-1, ZnSO4 g l-1, CuSO4 0.5 g l-1, CoSO4 0.5 g l-1 (Saibi et al 2011) then incubated at 30°C, 150 rpm for days Crude enzyme extract was obtained by centrifuging the liquid medium at 20 000 g, 4°C for 20 and collecting the supernatant for confirming the β-glucosidases activity Enzyme assay β-Glucosidases activity towards p-nitrophenyl-β-D-glucopyranoside (pNPG) was measured with use of amount Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 of p-nitrophenol (pNP) liberated from pNPG by using a calibration curve at 410 nm (Cai et al 1998) The reaction mixture contained 0.5 ml, mM pNPG in 50 mM sodium acetate buffer (pH 5.0) and an appropriately diluted enzyme solution 0.125 ml After incubation at 45°C for 10 min, the reaction was stopped after adding 1.25 mL, M Na2CO3, and the color that formed as a result of pNP liberation was measured at 410 nm One unit of β-glucosidases activity was defined as the amount of enzyme required to liberate μmol of pNP per minute under the assay conditions Specific activity is defined as the number of units per milligram of protein Cellobiase activity was assayed using cellobiose as substrate The enzymatic reaction mixtures (1 ml) containing 0.25 ml of enzyme solution and 0.75 ml of 0.5% cellobiose in 50 mM sodium acetate buffer (pH 5.0) were incubated for 30 at 50 C And then the mixtures were heated at 100 C for to stop the reaction The amount of glucose released was measured by Bio-sensor (Oji Scientific Instruments Co., Itd) One enzyme unit was defined as the amount of enzyme that produced μmol of glucose per minute Protein concentration determination Protein concentrations in the enzyme preparations were determined with application of the method of Bradford (Bradford 1976) with reference to a standard calibration curve for bovine serum albumin (BSA) Strain identification DNA extraction and PCR amplification from cultures Mycelia cultured on malt extract agar were harvested with a spatula, and DNA was extracted with use of a PrepManW Ultra Reagent (Life Technologies, Carlsbad, California, USA) ITS-5.8S rDNA (ITS) and the D1/D2 regions of LSU rDNA (LSU) were amplified with the KOD FX (Toyobo, Osaka, Japan), and with primers ITS5 (GGAAGTAAAAGTCGTAACAAGG) and NL4 (GGTC CGTGTTTCAAGACGG) (O'Donnell 1993; White et al 1990) The mixture was processed by following the manufacturer’s instructions of kit The DNA fragments were amplified in a T-gradient thermal-cycler (Biometra, Göttingen, Germany) Thermal-cycling program for LSU and ITS was: initial denaturation at 94°C for min, 30 cycles of denaturation at 98°C for 10 s, annealing at 56°C for 30 s, extension at 68°C for and a 4°C soak Amplified DNA was purified with use of the AgencourtW AMPureW Kit (Agencourt Bioscience, Beverly, Massachusetts, USA) DNA sequencing Sequencing reactions were performed with the BigDyeW Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA), and with primers Page of 13 NL1 (GCATATCAATAAGCGGAGGAAAAG) and NL4 (GGTCCGTGTTTCAAGACGG) for LSU on the Tgradient thermal-cycler (Biometra) This thermal-cycler program was employed: initial denaturation at 96°C for 1.5 min, 35 cycles of denaturation at 96°C for 10 s, annealing at 50°C for s, extension at 60°C for 1.5 and a 4°C soak Sequencing reaction products were purified with the AgencourtW CleanSEQW Kit (Agencourt Bioscience) and sequenced with the ABI PRISMW 3730 Genetic Analyzer (Applied Biosystems) Contiguous sequences were assembled with ATGC software (Genetyx, Tokyo, Japan) Phylogenetic analysis DNA was analyzed with use of CLUSTAL W (Thompson et al 1994) Based on the EF-1α sequence of Fusarium genus (O'Donnell et al 2012), phylogenetic tree was generated with use of the neighbor-joining algorithm in the MEGA ver5.0 Concordance of the EF-1a gene datasets was evaluated with the partition-homogeneity test implemented with MEGA (Tamura et al 2011), using 000 random repartitions The fungus was determined to be most closely related to Fusarium proliferatum by comparing it with related strains in GenBank And the NBRC deposition number is NBRC109045 Effect of different carbon sources on β-glucosidases production by F proliferatum The mycelium stored on PDA medium was transferred to new PDA medium in 9-cm diameter Petri dish and incubated at 30°C for days Once the fungus covered most of the PDA plate, agar plates with mycelium were transferred to a sterile blender containing 25 ml of sterile water and then homogenized for 30 s Ten ml of the fungal homogenate was used to inoculate 100 ml of liquid pre-cultures, pH 7.0 Liquid pre-cultures were made according to the modified Mandels medium with and without 0.69 g L-1 urea supplemented with 0.1% of yeast extract and 1% of glucose (Saibi et al 2011) After days, the mycelium homogenate made by a sterile blender was used to inoculate the modified Mandels medium which containing 2% carbon source with and without urea as following, wheat bran, corn stover, 1% wheat bran + 1% corn stover, bagasse, CMC, Avicel cellulose, sucrose, cellobiose, glucose, xylose, galactose and maltose β-Glucosidases production by F proliferatum in shaking flask batch cultures was carried out at 30°C and 150 rpm Samples were withdrawn at different times during 12 days, and then centrifuged at 20 000 g for 20 Supernatants as crude enzyme were assayed for β-glucosidases activity, determined for pH, and analyzed by zymogram Each culture was carried out in triplicate Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Page of 13 Electrophoresis and zymogram Zymography is an electrophoretic technique for detection of purified or partly purified β-glucosidase Zymography is based on SDS-PAGE that includes a substrate such as MUG or pNPG, which can be degraded by β-glucosidases The degradation product emits fluorescence or produces change of color during the reaction period However, this is not a practical method to assay β-glucosidases existing in the crude enzyme because various β-glucosidases existing in the crude enzyme caused overlapping fluorescence bands A modified method that combines effective isolation with identification was developed to overcome the limitation of zymogram in the application on crude enzyme Step1: add the loading buffer for SDS-PAGE to the crude enzyme solution that was produced by incubating F proliferatum in corn sotver + wheat bran based medium and glucose based medium, but the mix was not heated at a temperature of 100°C (Laemmli 1970) The mix of the crude enzyme and loading buffer was injected into the gel Each sample was injected into four different wells and then the electrophoresis was applied Step2: After the electrophoresis, the first column of each sample was cut out of the gel and then treated with Coomassie Brilliant Blue (CBB) staining The remaining gel was soaked in 20 mM, pH8.5 Tris–HCl buffer for two hours in order to remove SDS, so that the activity can be regained The buffer was replaced every 30 Step3: The first column that had been treated with CBB staining was used as a marker to cut the protein bands of the second column The protein bands cut out of the second column were soaked in 20 mM pNPG for 10 at a temperature of 45°C with the aim of active staining, and then 1.25 ml of M Na2CO3 solution were added If the color of the bands changes from colorlessness to yellow, it means that β-glucosidases exist in the bands Step4: Corresponding bands were cut out of the third and the fourth column based on positions of active bands of the second column The cuts containing β-glucosidases were soaked in acetate buffer (0.05 M, pH5.0), and were crushed and separated by centrifugation The supernatant was taken out and mixed with the same volume of loading buffer and then was analyzed with SDS-PAGE Protein was stained with silver stainIIkit (Wako Pure Chemical Industries, Ltd, Japan) Partial purification of β-glucosidase Fine and dried powder of ammonium sulfate was added, over ice, into the crude extract enzyme to 50% saturation Table Screening of microorganism with β-glucosidases production No Serial number 81 SIID11445 Cuc Phuong National Park Manihot esculenta wood chip +++ 82 SIID11446 Cuc Phuong National Park Rice straw + 83 SIID11447 Ba Be National Park Manihot esculenta wood chip + 84 SIID11448 Cuc Phuong National Park Soil around plant chip + 85 SIID11449 Cuc Phuong National Park Soil around plant chip ++ 86 SIID11450 Cuc Phuong National Park Soil around plant chip ++ 87 SIID11451 Cuc Phuong National Park Manihot esculenta wood chip + 88 SIID11452 Cuc Phuong National Park Jatropha carcass wood chip + 89 SIID11453 Ba Be National Park Jatropha carcass wood chip + 90 SIID11454 Ba Be National Park Jatropha carcass stems and leaves +++ 91 SIID11455 Cuc Phuong National Park Jatropha carcass stems and leaves ++ 92 SIID11456 Ba Be National Park Soil around plant chip +++ 93 SIID11457 Ba Be National Park Jatropha carcass wood chip + 94 SIID11458 Ba Be National Park Jatropha carcass wood chip ++ 95 SIID11459 Ba Be National Park Rice straw + 96 SIID11460 Ba Be National Park Coconut +++ 97 SIID11461 Ba Be National Park Rice straw + 98 SIID11462 Cuc Phuong National Park Coconut + +++: with the brightest fluorescence ++: with brighter fluorescence +: with bright fluorescence Sample site Source Fluorescence remarks Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 And then the mix was still stirring at 4°C for 30 After centrifugation (42 500 g, 60 min), supernatant was decanted and the precipitate was discarded Ammonium sulfate was added to bring the supernatant to 80% saturation The latter was stirred overnight at 4°C and then centrifuged again The precipitate was dissolved and dialyzed against 20 mM Tris–HCl buffer (pH 8.5) The dialyzed enzyme solution was centrifuged to remove the insoluble component and applied on the DEAE sepharose CL-6B column (1.5*20 cm) equilibrated with 20 mM Tris–HCl buffer (pH 8.5) The nonadsorbed protein fraction was eluted from the column with starting buffer (100 mL), and the adsorbed enzyme was collected through 5-stepwise elution chromatography (sodium chloride concentration: 0.1 M, 0.15 M, 0.2 M, 0.25 M and 0.3 M in the same buffer) There are two active peaks eluted from DEAESepharose CL-6B at about 0.15 M and 0.25 M NaCl The active fractions (0.15 M NaCl) were pooled and concentrated by a Centrifugal Filter Devices (Millipore Corporation Billerica, MA, USA), and then chromatographed separately on a superdex 75 column (1.5*60 cm) equilibrated with 20 mM Tris–HCl buffer (pH 8.5) The proteins were eluted with the same buffer at a flow rate of mL min-1 Page of 13 Results Screening of β-glucosidases-producing strain MUG released MU when MUG was catalyzed by β-glucosidases, and MU emitted fluorescence In order to screen the best strain for β-glucosidases production, firstly the potential strains were cultivated in medium that contained MUG Of these potential strains, strains showed the brightest fluorescence (Table 1) Next, these strains were prepared in a medium that contained 1% of corn stover and 1% of wheat bran for five days Of these strains, SIID 11460 showed the highest activity of β-glucosidases Therefore, SIID 11460 was selected for further research Strain identification The ITS1-5.8-ITS2 ribosomal RNA gene of SIID11460 was amplified with PCR for identification However, amplification showed no significant differences among the sequences of the PCR products generated with the internal transcribed spacer (ITS) primers Due to many fusaria within the Gibberella clade possess nonorthologous copies of ITS2, it can lead to incorrect phylogenetic inferences with use of ITS sequence identification (O'Donnell and Cigelnil 1997; O'Donnell et al 1998) Therefore, the elongation factor 1α (EF-1α) was Figure Phylogenetic tree based on EF-1α sequences of isolated strain SIID 11460 and other related species obtained from NCBI The phylogenetic tree was constructed by the neighbor-joining method using CLUSTAL W and MEGA ver5.0 Levels of bootstrap support were indicated at nodes The scale bar represents 0.005 nucleotide substitution per position Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Page of 13 Table The activity of β-glucosidases produced by F proliferatum growing on different carbon sources Carbon sources With urea (U/ml) Without urea (U/ml) Agricultural by-products Corn stover 0.90 ± 0.05 0.28 ± 0.04 Wheat bran 2.09 ± 0.13 1.26 ± 0.07 Baggase 1.32 ± 0.08 1.28 ± 0.08 Corn stover + wheat bran 3.31 ± 0.14 2.78 ± 0.10 0.37 ± 0.01 0.38 ± 0.08 Polysaccharides CMC Avicel cellulsoe 0.2 ± 0.004 0.10 ± 0.01 Disaccharides Sucrose 0.24 ± 0.04 Cellobiose 0.90 ± 0.03 Glucose 0.69 ± 0.02 Xylose 0.28 ± 0.08 Galactose 0.02 ± 0.001 Maltose 0.02 ± 0.002 Monosaccharides β-Glucosidases activity was determined based on pNPG as the substrate The different carbon sources were used at the concentration of 2% in modified Mandels culture medium Values are means ± SD of triplicate samples used for the identification of SIID11460 The EF-1α gene of SIID11460 was successfully amplified by PCR The fungal EF-1α gene was amplified from genomic DNA, and then purified, sequenced and analyzed with the BLAST program from NBRC The strain showed the highest identity (99.3 ~ 100%) with Gibberella intermedia (Fusarium proliferatum) Based on the EF-1α sequence of Fusarium genus (O'Donnell et al 2012), phylogenetic tree was built up Phylogenetic analysis indicated that SIID11460 and Gibberella intermedia NRRL 25103, Gibberella intermedia NRR52687 and Fusarium proliferatum NRRL 43545 belong to the same clade (Figure 1) Based on the comparison of the EF-1α gene sequences and the location of clade in the species complex of Gibberella fujikuroi (O'Donnell et al 1998; Nirenberg and O'Donnell 1998), the strain SIID11460 was identified as a strain of F proliferatum that belongs to Liseola section of the Fusarium genus (Nelson et al 1983) and its teleomorph is Gibberella intermedia SIID11460 was named as F proliferatumNBRC109045 β-glucosidases production by F proliferatum in various carbon sources Various carbon sources, not only agricultural byproducts and polysaccharides but also mono- and disaccharides were tested for β-glucosidases production by F proliferatum with and without urea for 10-day cultivation (Table 2) All substances with urea addition induced β-glucosidases production at different levels When pNPG was used as substrate to measure activity of β-glucosidases, the activity reached the highest level of 3.31 ± 0.14 U/ml with use of corn stover + wheat bran as carbon source The activity level was still as high as 2.09 ± 0.13 U/ml when wheat bran was used as carbon source An activity of 0.69 ± 0.02 U/ml was assayed when the glucose was used as carbon source even though glucose is regarded as a universal repressor of hydrolases The activity level produced with use of glucose as carbon source was a little bit below the activity level produced with use of cellobiose as carbon source When disaccharides and monosaccharides were used as the sole source of carbon at pH 7.0 without urea, no activity of β-glucosidase was detected even extending the period of cultivation to 25 days Only agricultural byproducts and polysaccharides at pH 7.0 without urea addition induced β-glucosidases production The variation of pH before and after culturing was expressed in Table Before cultivation of F proliferatum, the pH of mediums was adjusted to 7.0 Ten days later, the pH values of glucose or cellobiose based mediums without urea addition dropped to approximately 2.5; the pH values of glucose or cellobiose based mediums with urea addition hardly changed; the pH values of corn stover + wheat bran based mediums with and without urea addition were 7.1 and 6.0, respectively, after 10-day cultivation It is reported that the biosynthesis of β-glucosidases is greatly influenced by pH (Tangnu et al 1981; Desrochers et al 1981) For F proliferatum in this study, low pH of the glucose or cellobiose based mediums cut production of β-glucosidases But addition of urea halted reduction in pH of glucose or cellobiose based mediums When F proliferatum grew in corn stover + wheat bran based medium, the pH decreased slightly Therefore, whether adding urea to corn stover + Table The pH of mediums in which F proliferatum grew for 10 days With urea addition Glucose Without urea addition Before cultivation pH After cultivation pH BGL activity (U/ml) Before cultivation pH After cultivation pH BGL Activity (U/ml) 7.0 6.5 ± 0.2 0.69 ± 0.02 7.0 2.5 ± 0.2 cellobiose 7.0 6.5 ± 0.2 0.90 ± 0.03 7.0 2.6 ± 0.3 Corn stover + wheat bran 7.0 7.1 ± 0.1 3.31 ± 0.14 7.0 6.0 ± 0.1 2.78 ± 0.02 BGL: β-glucosidases Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Page of 13 wheat bran based medium did not affect production of β-glucosidases evidently Thus, the addition of urea might have the ability to promote the production of β-glucosidases, especially in mono and disaccharides To make sure of the function of urea, a comparative test was carried out Figure indicated the time course of β-glucosidases production by F proliferatum using different carbon sources with and without addition of urea F proliferatum started to produce β-glucosidases on the 8th day after incubating in glucose or cellobiose based medium with urea addition (Figure 2-a) According to the time course for β-glucosidases production, the same amount of urea was added to the glucose and cellobiose based mediums on the 8th day after incubating, respectively Then the samples were taken out every days to determine the activity of β-glucosidases and pH However, F proliferatum did not produce β-glucosidases and the pH of the mediums was kept at about 2.5 The results indicated that there was no relationship between addition of urea and halting reductions in pH of glucose or cellobiose based medium β-glucosidase activity (U/ml) a 3.5 2.5 1.5 0.5 0 Time (day) 10 12 3.5 100 90 2.5 80 Activity retention (%) β-glucosidase activity (U/ml) b Figure indicated that the glucose tolerance of the β-glucosidases produced by F proliferatum growing in varied carbon sources based mediums Supplementation of glucose in the substrate resulted in severe reductions in β-glucosidases activity On the other hand, β-glucosidases produced by F proliferatum growing in corn stover + wheat bran based medium had higher tolerance to glucose compared to that in glucose or cellobisoe based medium β-Glucosidases produced with use of different carbon sources have different level of tolerance to the glucose β-Glucosidases may be classified into three groups on the basis of substrate specificity (1) Aryl β-glucosidases exclusively hydrolysing or showing a great preference towards aryl β-glucosides; (2) cellobiases hydrolysing cellobiose and small oligosaccharides and finally (3) the members of the third group, termed as broad-specificity β-glucosidases, that act on both substrates (aryl-β-glucosides, cellobiose and cellooligosaccharides) and are the most commonly observed group in cellulolytic microbes (Patchett et al 1987) The hydrolysis capacity of β-glucosidases produced by F proliferatum growing in corn stover + wheat bran based medium and glucose based medium were tested on cellobiose (0.5%) After 30 min, aliquots were taken out and their glucose contents were determined by Bio-sensor Based on the substrate of cellobiose, the activities of β-glucosidases produced by F proliferatum growing in corn stover + wheat bran based medium and glucose based medium were 426 U/ml and 187 U/ml, respectively According to the results mentioned above and those in Table 2, β-glucosidases produced by F proliferatum grew in corn stover + wheat bran based medium and glucose based medium belongs to the third group of β-glucosidases, due to the capacity of β-glucosidases to hydrolyze cellobiose and pNPG 1.5 0.5 0 Time (day) 10 12 Figure Time course of β-glucosidases production by F proliferatum using different carbon sources a: with addition of urea b: without addition of urea Corn stover + wheat bran (⋄), bagasse(□), CMC(5), cellobiose(Χ), glucose(*), and xylose (○) were used individually, at the concentration of 2% in the modified Mandels medium Samples were withdrawn every two days during 12 days 70 60 50 40 30 20 10 0 50 100 150 200 250 Glucose concentration (mM) 300 Figure The glucose tolerance of the β-glucosidases produced by F proliferatum growing in varied carbon sources based mediums Corn stover + wheat bran (5), cellobiose (□), and glucose (○) Values are means ± SD of triplicate samples Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Differential expression of β-glucosidases in response to carbon sources Zymogram analysis was used to assay the β-glucosidases produced by F proliferatum that grew in corn stover + wheat bran based medium and glucose based medium Figure (See legend on next page.) Page of 13 When zymogram analysis was used to detect different β-glucosidases existing in the crude enzyme, the exact number of the fluorescence bands could not be identified because the fluorescence bands overlapped each other, and it was also difficult to get clear pictures Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Page of 13 (See figure on previous page.) Figure Schematic of the modified zymogram a: add the mix of loading buffer and crude enzyme solution to the gel But the mix was not heated at 100°C Blue: crude enzyme from glucose based medium; Green: crude enzyme from corn stover + wheat bran based medium; Red: loading buffer only b: after electrophoresis, the first column of each sample was cut out c: the first column of each sample was stained with CBB d: the remaining gel was soaked in Tris–HCl buffer to remove SDS e: the first column after CBB staining was used as a marker to cut the protein bank of the second column f: the protein bank cut of the second column was soaked in pNPG for active staining g: after adding Na2CO3, the band coming from the second column kept colorlessness h: the color of the band from the second column changed from colorlessness to yellow following addition of Na2CO3 i: according to the position of active band of the second column, cut the corresponding bands of the third and fourth column j: protein coming from the bands of the third and fourth column was injected to the gel for SDS-PAGE following a series of treatments Therefore, we modified the zymogram method and usefully applied the modified method to prove a differential expression pattern of β-glucosidases produced by F proliferatum that grew in the carbon sources (Figure 4) After the electrophoresis, the first column of each sample was cut out of the gel and then treated with Coomassie Brilliant Blue (CBB) staining Figure 5-a shows bands of proteins that existed in the crude enzyme growing in glucose based medium and bands of proteins that existed in the crude enzyme growing in corn stover + wheat bran based medium Based on the stained bands of the first column, the correspondent gel bands on the second column of the same sample were cut as narrow as possible and these cuts were separately incubated in pNPG for 10 Actually, bands Glu2,Glu3, Glu4,Glu7,CW2,CW4,CW5 changed to yellow That proved existence of β-glucosidase activity Among these stained bands, colors of band Glu7 and CW2 were the most visible The position of band Glu2 at the gel corresponded to that of band CW2, band Glu3 matched with band CW4, and band Glu4 was corresponding to band CW5 Corresponding band of Glu7 was not found at the CW gel Band CW7 was cut out of CW gel based on the position of band Glu7 on the assumption that the same β-glucosidases would be produced by F proliferatum that grows in different carbon sources The cut was treated for activity staining but no change of color was observed It indicated that the cut did not contain any β-glucosidase Subsequently, the bands with β-glucosidase activity on the second column were used as markers to cut the corresponding bands out of the third and fourth column of the same sample as narrow as possible The cuts were soaked in acetate buffer (0.05 M, pH5.0) to recover the protein of β-glucosidase containing in the gel and treated with SDS-PAGE and then with silver staining Figure 5-b indicates the results of the SDS-PAGE However, the amount of proteins existing in bands Glu2,Glu3, Glu4,CW4 and CW5 was too low to be visible after the SDS-PAGE In all, at least four different β-glucosidases were produced by F proliferatum growing in glucose Figure Zymogram demonstrated that F proliferatum expressed differentially in response to various carbon source at 2% (w/v) a: Coomassie staining of SDS-PAGE of crude enzyme b: Silver staining of the SDS–PAGE Glu, glucose; CW, corn stover + wheat bran; M, molecular weight marker (kDa); B, loading buffer only Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Page 10 of 13 based medium and at least three different β-glucosidases were produced by F proliferatum growing in medium of corn stover + whea bran β-Glucosidase with the molecular weight of approximate 46 was produced in glucose based medium only Therefore, we came to a conclusion that different β-glucosidases can be produced by that grows in different carbon based mediums Partial purification of β-glucosidase The partial purification process was summarized in Table In the initial step of purification with ammonium sulfate fractionation, about 70% of total β-glucosidase activities could be recovered in the fraction of 50–70% ammonium sulfate saturation with a purification of 3.3 times In the second step, ion-exchange chromatography on DEAE-Sepharose CL-6B was performed using five concentration of sodium chloride for elution In this step, greater purity was realized since most of the contaminating protein was removed β-glucosidase was eluted from the ion exchanger at the sodium chloride concentration of 0.15 M, as one broad peak About 32% of total β-glucosidase activities could be recovered Accordingly, β-glucosidase was purified 9.2 times In the third step, active fraction (0.15 M NaCl) gained from DEAESepharose CL-6B was applied on Superdex 75 column About 16% of total β-glucosidase activities could be recovered As a result, β-glucosidase was purified 18.0 times After all these steps, we got β-glucosidase that had a specific activity of 287.7 U/mg based on pNPG and 400 U/mg based on cellobiose The results pointed out that β-glucosidase produced by F proliferatum that grows in corn stover + wheat bran based medium has high catalytic efficiency (Table 5) There were two major bands on the SDS-PAGE of the active peak from Superdex 75 Compared the location band of CW2 that came from the modified zymogram and active peak from superdex75 (Figure 6-c), we can get the conclusion that the band on the top of lane on Figure 6-c is the β-glucosidase we need to purify Discussion Cellobiose was considered as an inducer of cellulase which includes β-glucosidases (Mandels and Reese 1957) However, the amount of β-glucosidases when F proliferatum grew in cellobiose based medium was Table Specific activity of purified β-glucosidase from various sources Strain Specific activity (U/mg) Reference Rhizomucor miehei (NRRL 5282) 62 (Krisch et al 2012) Candida peltata (NRRL Y-6888) 108 (Saha and Bothast 1996) Daldinia eschscholzii 78 (Karnchanatat et al 2007) Stachybotrys microspora 20 (Saibi and Gargouri 2011) Thymepkilic anaerobic bacterium 149 (Patchett et al 1987) Aspergillus niger (NIAB 280) 42 (Rashid and Siddiqui 1997) Xylaria regalis 23 (Wei et al 1996) Trichoderma sp 214 (Fadda et al 1994) Aspergillus niger (CCRC 31494) 199 (Yan and Lin 1997) Fusarium proliferatum (NBRC 109045) 288* This study *partially purified less than that in corn stover + wheat bran based medium When compared the yield of β-glucosidases in cellobiose based medium with that in corn stover + wheat bran based medium, F proliferatum grew in cellobiose faster than that in corn stover + wheat bran (data not shown) This proved that cellobiose is an excellent growth substance for and is rapidly consumed, whereas corn stover + wheat bran is a relatively poor growth substance and is slowly consumed The same phenomenon was observed by (Mandels and Reese 1960) They held the opinion that the inhibitory effect of cellobiose on β-glucosidases production seems to be related to rapid metabolism of the cellobiose Wheat bran that contains significant quantities of starch, protein and so on is a rich source of nutrients and could promote growth and enzyme production of fungus Corn stover that is mainly composed of lignocellulose is a very common and cost-free agricultural product Supplementation of the mixture of wheat bran and corn stover resulted in a significant increase in β-glucosidases activity when compared to individual application The likely reasons for the result were that wheat bran provided F proliferatum with adequate nutrition at the early growth stage and made the strain grow fast After nutrition contained in wheat bran ran out, F proliferatum Table Summary of the purification steps of the β-glucosidase produced by F proliferatum growing in corn stover + wheat bran based medium Step Total activity (U) Total protein (mg) Specific activity (U/mg) Purification factor Crude extract 150 9.38 16.0 1.0 Recovery (%) 100 (NH4)2SO4 106 1.98 53.5 3.3 70.4 DEAE Sepharose CL-6B 48.6 0.33 148 9.2 32.3 Superdex75 23.8 0.08 288 18 15.8 Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 Figure SDS-PAGE analysis (10% polyacrylamide) of protein sample from each step of the partial purification a-(1), molecular weight markers; a-(2), crude protein extract; b-(1), the active peak from DEAE sepharose CL-6B; b-(2), molecular weight markers; c-1, the protein from CW2 band of the modified zymogram; c-(2), the protein from the active peak of Superdex75; c-(3) molecular weight markers; c-(4), loading buffer only The protein showed on A and B was stained with CBB R-250 The protein showed on C was with silver staining could hardly get required nutrition from corn stover and became starve and then produced a huge amount of β-glucosidases F proliferatum did not produce β-glucosidases in glucose or cellobiose based medium without addition of urea (Table 2) And it has been proved that there was no direct relationship between addition of urea and halting reduction in the pH of glucose or cellobiose based medium Emergence of the phenomenon prompted us to ponder a problem that how the addition of urea contributed to β-glucosidase production in glucose or cellobiose based medium This is probably due to the metabolites produced by F proliferatum growing in glucose or cellobiose based medium or the derivatives of the components containing in the glucose or cellobiose Page 11 of 13 based medium The metabolites or derivatives produced by the fungus would reduce the pH of the glucose or cellobiose based medium Low pH of the glucose or cellobiose based medium, in turn, cut the production of β-glucosidases But addition of urea at the beginning of cultivation can cut the production of the metabolites or derivatives That, in turn, halted reduction in pH of glucose or cellobiose based medium However, adding urea to the glucose or cellobiose based medium after 8-day cultivation cannot damage the metabolites or derivatives produced in large quantities during incubation In this case β-glucosidases still cannot be produced by F proliferatum even addition of urea The possible reasons for slight decrease in pH of the corn stover + wheat bran based medium are because the metabolites or derivatives were not produced by F proliferatum, or only tiny amount of the metabolites or derivatives was produced Therefore, urea addition did not affect the production of β-glucosidases produced by F proliferatum growing in corn stover + wheat bran based medium significantly β-Glucosidases produced by F proliferatum in different carbon sources based mediums expressed varied glucose tolerance (Figure 3) (Isorna et al 2007) purified a β-glucosidase, named as BglB, produced by P polymyxa and obtained the crystallographic structure of the BglB with glucose In this structure, the ring of glucose resided in the active site, through the interactions with nine amino acids of BglB Of the nine residues, seven were involved in intermediate binding to glucose directly, while the other two, Trp412 and His181, indirectly binding to glucose The seven directly interacting residues were found to conserve among different β-glucosidases belonging to GH1, whereas Trp412 and His181 in BglB are fairly variable The two variable residues were assumed to play important roles in glucose tolerance It has been proved that the 184th residue of β-glucosidase BglB plays an important role in glucose tolerance (Liu et al 2011) Glucose acts as an inhibitor by competing with the substrate in binding to the enzyme (Fang et al 2010) But the mechanism of β-glucosidase tolerance to glucose is still unclear Presumably the cause is that there are variable special residuals on active site of β-glucosidases by F proliferatum in different carbon sources based mediums These residuals are not only the binding site of glucose but the binding site of the substrate, so changes of the special residuals cause difference in degree of bond to glucose That, in turn, makes difference in level of tolerance to glucose We reported the modified zymogram method that is a combination of the technology zymogram, gel purification and SDS-PAGE to prove that F proliferatum could express varied β-glucosidases in respond to varied carbon sources The approach described in the paper overcomes the disadvantages of applying crude enzyme on Gao et al AMB Express 2012, 2:49 http://www.amb-express.com/content/2/1/49 zymogram and combines effective isolation with identification assay Competing interests The authors declare that they have no competing interests Author details Department of Agro-Environmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan 2Center of Biotechnology, Vietnam National University, Hanoi, Vietnam 3Department of Biotechnology, National Institute of Technology and Evaluation, Tokyo, Japan 4Advanced Technology Laboratories IDEMITSU KOSAN Co., Ltd, Chiba, Japan Received: 23 August 2012 Accepted: 29 August 2012 Published: 14 September 2012 References Adsul MG, Bastawde KB, Varma AJ, Gokhale DV (2007) Strain improvement of Penicillium janthinellum NCIM1171 for increased cellulase production Bioresour Technol 98:1467–1473 Banerjee S, Mudliar S, Sen R, Giri B, Satpute D, Chakrabarti T, Pandey RA (2010) Commercializing lignocellulosic bioethanol: technology bottlenecks and possible remedies Biofuels Bioprod Bioref 4:77–93 Beguin P, Aubert JP (1994) The biological degradation of cellulose FEMS Microbiol Rev 13:25–28 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72:248–254 Cai YJ, Buswell JA, Chang ST (1998) β-Glucosidase component of the cellulolytic system of the edible straw mushroom, Volvariella volvacea Enzyme Microb Technol 22:122–129 Crout DH, Vic G 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production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest AMB Express 2012 2:49 Submit your manuscript to a journal and benefit from: Convenient online submission Rigorous peer review Immediate publication on acceptance Open access: articles freely available online High visibility within the field Retaining the copyright to your article Submit your next manuscript at springeropen.com ... pattern of β-glucosidases produced by F proliferatum that grew in the carbon sources (Figure 4) After the electrophoresis, the first column of each sample was cut out of the gel and then treated... Gao et al.: The production of β-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest AMB Express 2012 2:49 Submit your manuscript to a journal and benefit from: Convenient... produced by F proliferatum, or only tiny amount of the metabolites or derivatives was produced Therefore, urea addition did not affect the production of β-glucosidases produced by F proliferatum